Identification of cell-of-origin subtypes and a wound healing response signature in inflammatory breast cancer

Poster Sessions

Thursday, 23 March 2006

used for this purpose The objective of this study was to test the ability of intraoperative touch imprint cytology (IC) to predict metastatic disease on SLN Design: SLN were recaived fresh and examined grossly, when less than 05crn in size were bisected and when more than 0 5 c m in size were serially sectioned at 2 mm intervals along the long axis Each surface of the sections were touched on the glass slide, stained by H&E. Results of IC were compared with results of section permanents, wich were analyzed with 10 serial levels stained with H&E and one level stained with cytokerafin AE 1f AE 3. Sensitivity (So), specificity (Sp), positive and negative predictive value (PPV & NPV) and accuracy were calculated for all metastases (macro & micrometastases), micrometastases, macrometastases. False negatives were rescreened. Results: We analyzed 179 SLN from 110 patients The comparison between IC and definitive results of the SLN (including macro & micrometastases) showed 139 (77 65%) true negative impdnts. 28 (15 64%) true positive imprints There were not false positive imprints, there were 12 (6 70%) false negative imprints False negative imprints were 6 macrometastases (mean size metastases 5mm. range 3 7mm). 3 micrometastases (mean size metastases 1 6 mm. range 2-1 5mm) and 3 isolated tumour cells. Rescreening of the false negative imprints showed 10 negative impnnts, one imprint with two diagnostic groups of cells and one imprint with multiple diagnostic groups of cells. So, Sp, PPV, NPV, Acc for all metastases, micrometastases, macrometastases are shown in the table.

All metastases Micrometastases Macrometastases

Se

NPV

Sp

PPV

Acc

70% 73 60% 82 35%

92.05% 93 37% 96 02%

100% 100% 100%

100% 100% 100%

93.29% 94 41% 96 64%

Conclusions: The majority of macrometastases can be detected by IC however IC fails to detect most micrometastases False negative imprints fur macrometastases are mainly due to sampling error. The high Sp, PPV and preservation of the arquitecture of the lymph node for histopathologic examination are the major advantages of IC for intraoperative evaluation of SLN.

Thursday, 23 March 2006

16:00-16:45

POSTER SESSION

Tumour biology and immunology

125

and 2 remains virtually unknown to date Here. we show Bexl and 2 classify a novel subtype of ER positive breast tumors which respond better to tamoxifen therapy We demonstrate Box2 is part of estrogen response and NGF/NF-~B pathways with anti-apoptotic function in breast cancer NF-~B activity has recently gained much attention in the development of hormone refractory breast cancer and Box2 can potentially be applied as an activity marker or therapeutic target within this pathway. The findings reported here show Box1 and 2 are novel breast cancer related genes and significantly advance our understanding of NGF/NF ~B pathway with potential clinical implications.

268 Poster The prognostic significance of inflammation in invasive carcinoma of the breast A. Lee I . I. Ellis I . M. Mitchell 2, R. Blarney 2, C. Elston I . ~Notttngham City

Hospital Department of Histopathoiogy, Nottingham, Ur~itedKingdom, 2NotUngham City Hospital, Department of Surgery, NotUrtgham, Urtited Kingdom The prognostic significance of inflammation in invasive carcinoma of the breast is controversial with previous studies producing conflicting results The predominant pattern of inflammation is a diffuse infiltrate of T calls and macrophages in the stroma between carcinoma cells Pedvascular and perilobular clusters of B and T cells are less prominent The calls necessary for a call-mediated immune response are often present, but there is evidence that their function is impaired Inflammatory cells may also stimulate tumour growth by release of proleolytic enzymes or angiogenic factors. 1599 patients aged less than 71 years with operable invasive carcinomas, diagnosed frum 1974 to 1988, with median fellow up 9.4 years were studied. No patient received adjuvant systemic treatment. An overall assessment of the intensity of lympho hisfiocyfic inflammation was made on haematoxylin and eosin sections by one observen Inflammation was associated with higher grade, ductal and medullary histological types, tumour size and inversely with patient age. On univanate analysis patients with turnouts with marked or moderate inflammation had a better survival than patients with turnouts with absent or mild inflammation (P 0 04) On multivariate analysis survival was associated with inflammation (relative risk 0 61 (95% confidence intervals 0 47 to 0 79). P 0 0002) in addition to lymph node stage, histological grade, turnout size, vascular invasion and turnout type: survival was not related to patient age or oestrogen receptor status This study suggests that the anti4umour effects of inflammation predominate over the pro tumour effects. Critical review of previous large studies with assessment of histological grade and mulfivanate analysis shows that the majority find prominent inflammation is associated with a better prognosis, consistent with the present study. These results support further studies trying to harness the immune response in the treatment of breast cancer.

267 Poster Box2 identifies a novel subtype of breast cancer associated with estrogen-response and NGF/NF-KB pathway

269 Poster Identification of cell-of-origin subtypes and a wound healing response signature in inflammatory breast cancer

A. Naderi ~, A. TeschendorlI4 , J. Beigel ~, M. Cariafi 1 , I. Ellis 2, J. Brenton 1, C. Caldas ~. ~HutchisontMRC Research lnsbtute, Oncology, Cambridge,

S. Van Laere 1, G. Van den Eynden 1, I. Van der Auwer'a 1, V. Huygelen ~, H. Elst ~, C. Colpaert 1, P. van Dam 1, E. Van Marck ~, P Vermeulen 1, L. Dirix 1. ~Transta~onalCancer Research Group, (Lab Pathology University

Ur~ited Kingdom, 2Nottingham University, Histopathology, Nottingham, Urtited i'Ortgdom Background: Heterogeneity of breast cancar is a significant challenge in diagnosis and therapy of the disease Despite advancements in the molecular profiling of breast cancer, there are still only three known molecular subtypes which can he used as classifiers; ER+. ER . and ERBB2+ Better molecular classification of breast cancar can improve our understanding of the disease and potentially lead to the discovery of novel therapies. Methods: In this study we performed microarray expression analysis of 135 breast tumors to identify novel classifiers in breast cancer. In addition, we evaluated clinical and biological relevance of our findings. Results: We identified Box1 and 2 genes as novel classifiers of breast cancen Overexpression of these genes was present in 15% of samples and was associated with estrogen-response and apoptotic function We showed Bex2 expression is necessary and sufficient for NGF anti-apoptotic activity Moreover, Box2 induction is mediated through p75NTR and located upstream of NF-~B Furthermore, estrogen induces Box2 in a time and dose dependent fashion and Bex2 is necassary for estrogen mediated anti-apoptotic activity We also found cases with Box2 overexpression responded better to tamoxifen therapy and proved the interaction between Box2 and tamoxifen activity in breast cancer cells. Conclusion: Although the importance of NGF/Bex3fNF ~B pathway is well known in neural tissues, NGF has recently been implicated in pathogenesis of breast cancer as well. Importantly, the function of Box1

of Antwerp and Oncology Center, GH Sir~t-Augustinus), Wilrijk, Belgium Introduction: Recently. gene expression studies demonstrated the significance of different biological breast cancar subtypes with regard to prognosis and treatment In this study we tested to what extent these subtypes contribute to the specific inflammatory breast cancer phenotype (IBC) Materials and Methods: The presence of different cell oPorigin subtypes (Perou et al.) and of a wound healing signature (Chang et al.) was analyzed in gene expression data sets from 16 IBC and 18 nlBC specimens. A set was compiled of genes being part of respectively the intnnsic gone set (Perou et al.) and the wound healing response signature (Chang etal.) which were also present on the cDNA microarrays used to compare IBC and nlBC specimens (Van Laere et al). 144 and 98 genes were selected from both gene lists These gene lists were then tested for performance in the original data sets Next. cantroids for each cell-of, origin subtype and for the quiescent and activated fibroblast signature were calculated These centroids were than used to classify our specimens For the cell-of-origin subtype classification, the robustness of the taxonomy was confirmed using an alternative data set of 141 genes related to the cell-of-origin subtypes Contribution of each of the cell-of-origin subtypes to the IBC phenotype was tested by principle component analysis (PCA). Results: The perfurmance of the selected data sets was 84% and 100%, respectively. 8f16 IBC specimens belonged to the combined Basal like and ErbB2 overexpressing duster, compared to only 3fl 8 nlBC specimens

126

Thursday, 23 March 2006

(p 0 037) The dassification was in good agreement with IHC data for ER and CK5f6 PCA revealed that only 35% of the differences between IBC and nlBC can be explained by the presence of the cell-of-origin subtypes 12/16 IBC specimens correlated with the quiescent fibroblast signature centroid compared with 5/18 nlBC specimens (p 0006) When only significant correlations were taken into account. 6f6 IBC and 3/8 nlBC specimens correlated with the quiescent fibroblast signature centroid (p = 0.03). These data are currently confirmed using real time qRTPCR. Discussion: 9 data sustain our previous findings that IBC and nlBC are two distinct biological entitities. Different cell oFongin subtypes in IBC were identified, but cannot fully explain the specific phenotype. Other processes must determine the biology of IBC, as shown by the differential expression of the wound healing response signature. 270 Poster Effect of Ubolone on breast cancer cell proliferation in postmenopausal ER+ patients: results from a double-blind, placebo-controlled, randomized dinical trial (STEM) E Kubista 1 . M Dowsett 2. J Foidart s. K Polhlodek 4. R Serreyn 5. M Nechushkin e, A Manikhas 7. V Semiglazov a, A Willemsen 9, J Planellas Gomez 1~ ~Medical Ur~iverdty of Vienna, Dept of Senology, Vienna,

Austria, 2Royal Marsden Hospital Dept of Academic Biochemistry, London, Umted K~ngdom, 3Untverstty of Liege, Department of OBtGYN, Doge, Belgium, 4University of Bratidava, Dept of OB/GYN, Brattslava, Sfovatqa; UmversJty of Ghent, Dept of OBt GYN, Ghent, Belgium, ~Cancer Research Center, Moscow, Rus~a, 7St Patersbog City C#mcal Oncology Dispensary, St Petersburg, Russia, 8Research Irtstitute of Oncology, St Petersburg, Russia, ~Organon NV, Dept of Biometrics, Oss, The Netherlands, ~~Organon NV, Dept of Clirtical Development, Oss, 77~eNetherlands Experimental design: Postmenopausal women with stage I or I1. ER+ primary breast cancer, were randomly assigned to 14 days of placebo or 2 5 mgfday tibolone Core biopsies of the primary tumor were obtained before therapy, and a representative sample of the excised tumor was obtained from the operative specimen alter treatment. For each patient, Ki67 and, apoptosis index were analyzed in both baseline and the corresponding post~reatment specimen. Results: Of 102 enrolled patients, 95 had evaluable data. Baseline characteristics were comparable between both treatment groups. Most breast cancer cases were invasive (99%). stage I or II (42% and 50% respectively) and ER+ (99%) Median intratumoral Ki67 expression at baseline was 13 0% in the tibolone group and 178% in the placebo group, and decreased to 12 0% after 14 days of tibolone while increasing non-significantly to 19 0% in the placebo group Similarly. no significant differences were observed between the treatment groups when the median baseline apoptosis index (1 4% in both groups) was compared to the corresponding post-therapeutic indices of 1 6% (tibolone) and 1 7% (placebo). No differences between tibolone and placebo were observed with respect to the incidence of adverse effects. Conclusion: 2.5rag/day Ubolone given for 14 days has no significant effect on tumor cell proliferation and apoptosis in ER+ tumors. 271 Poster Expression of FOXP3 and vascular endothelial growth factor in human breast cancer: its correlation with angiogenesis and disease progression S Gupta 1, K Joshi 2, J Wig ~. S Arora ~ ~Post Graduate Institute

of Medical Educatior~ and Research, immunopathology, Chandigart~, thdJa; 2Post Graduate Institute of Medical Education and Research, Htstopathofogy, Chandtgarh, Indra; ~Post Graduate thsbtute ot Medtcal Education and Research, General Surgery, Chandtgarh, thdta Background and Objective: 9 Forkhead/winged helix tnanscrip tion [actor FOXP3 is positively associated with the induction of CD4+CD25+CD45RA - 9 regulatory cells, which have a suppressive effect on the effector 9 cells. To determine whether FOXP3 might be involved in the progression of breast carcinoma, we measured the expression of FOXP3 in infiltrating breast carcinoma along with their corresponding normal breast tissues and a smaller number of ductal carcinoma in situ (DCIS) specimens and correlated it with the expression of Vascular Endothelial Growth Factor (VEGF. an invasogenic and angiogenic growth factor) and intratumoral microvessel density (IMD. a prognostic marker for angiogenesis) Methods: FOXP3 and VEGF mRNA expression was semiquantitated by R~PCR assay in 20 biopsies of infiltrating breast carcinoma, ,5 biopsies of DCIS along with 20 biopsies of normal breast tissues and analyzed their correlation with each other and with the IMD. determined by

Poster Sessions immunohistochemical staining using anti-CD34 antibody We also analyzed whether FOXP3 mRNA expression correlated with other pathological variables like tumor size. histological grade and lymph node status, the prognostic indicators of breast carcinoma Results: Invasive cancers had nearly three times greater FOXP3 mRNA expression than did ductal carcinoma in situ and nearly eight times greater than normal tissue and the difference was statistically significant (P < 0.0,5; P < 0.02. two tailed t test respectively). There appeared to be a trend towards increasing FOXP3 mRNA expression with increase in tumor size, with the larger tumors (~> 2.0 cm) having approximately two fold higher FOXP3 expression than the smaller tumors (< 2.0 cm), although the difference was statistically insignificant. FOXP3 expression was also found to be increased in higher grade tumors (0.05

Cancer Research Group, Laboratory of Pathology University ol Antwerp, Ortcology Centre, GH St-Augusb?lus, Antwerp, Belgium Angiogenesis is a fundamental process in turnout growth and metastatic dissemination. 9 number of circulating endothelial cells (CECs) in penpheral blood (PB) of patients with cancer reflects the amount of proceeding angiogenesis and can therefore be used as a surrogate marker to monitor antiangfegenic therapy. 9 standard quantification method of CECs is currently based on a complex four-color flow cytometry Real-time RT-PCR technology to quantify EC-specific mRNA in PB samples has been shown to be a promising alternative approach This study aimed to compare mRNA expression levels of EC-spedfic markers (CD146 and VE-cadherin) in PB of healthy volunteers and patients with breast cancer using real-time RT-PCR PB samples have been collected from 18 healthy volunteers and 18 metastatic breast cancer patients. RNA was subsequently isolated with the PAXgene Blood RNA isolation kit. Real4ime PCR analysis was performed with primers and 9 probes fur both CD146 and VE cadherin mRNA. Ct values were normalised for beta actin mRNA expression and gone expression levels were calculated relative to a reference sample (RGE). VE cadhenn mRNA was increased in patients with breast cancer in comparison to healthy volunteers: the median VE cadherin mRNA expression level in PB of healthy volunteers was 1 20 (range 0 50 4 18): this was 2 45 (range 0 69 25 80) for patients with breast cancer (p 0 040) However. the difference in CD146 mRNA expression levels between healthy volunteers and patients with breast cancer did not reach statistical significance: the median CD146 mRNA expression level in PB of healthy volunteers was 0 037 (range 0 020 0 058): this was 0 058 (range 0013 0488) for patients with breast cancer (p 0077) CD146 and VE cadherin mRNA expressions were significantly correlated (r= 0.401. p 0 017) A cut-off value was determined as the 95 th percentile of the RGE values of the healthy volunteers: this value was 0 058 for CD146 and 4 184 for VE-cadherin mRNA 9 out of 17 patients with breast cancer had a RGE of CD146 above the cut-offvalue:for VE-cadherin 7 out of 18 patients with breast cancer had increased RGEs Our preliminary results suggest that the quantitative evaluation of ECspecific mRNA by re.al-time RT-PCR could indeed be a promising tool to monitor the elficfency of antiangiogenic therapy in patients with breast cancer but a larger study population and a comparison with flow cytometry is necessary to confirm this. These studies are ongoing. 273 Poster Local aromatase and sulfotransferase protein expression in malignant breast tumors vs adjacent and distant breast tissue C. Sin.qer~. A. Ffek Rotter1, D. Gschwantler Kaulich 1, G. Hudelist ~. R. Mueller 1, K. Czerwenka 2, E. Kubista 1. ~MedJcaf Umverstty o1 Vienna,

Dept ot Senoiogy, Vienna, Austria; #Medical UmversJty of Vienna, Dept of Gyr~ecopathology, Vierma, Austtfa The suppression of local estrogens levels is of key importance in the treatment of ER positive breast cancer. Most endocrine strategies now